3-Hydroxy fatty acids (3-OH-FAs), derived from Gram-negative bacterial outer membranes, have received recent attention for their potential as new terrestrial pH and temperature proxies for palaeoclimate studies. However, it is not known whether 3-OH-FA based proxies can be developed and applied to the marine environment. Here we analyze 3-OH-FAs from a latitudinal transect of marine surface sediments from the North Pacific Ocean (12°N to 61°N with a annual mean sea surface temperature (SST) range of 28.1–1.3 °C). The results show that distributions of 3-OH-FAs in marine sediments yield overall higher abundances of anteiso 3-OH-FA homologues compared to soils. Furthermore, 16S rRNA gene sequencing of the marine sediments and soils shows the Gram-negative bacterial community is dominated by Proteobacteria (ca. 94%) at the phylum level. In contrast, in regional soils the Gram-negative bacterial community is more diverse with significant contributions from Proteobacteria (ca. 50%), Acidobacteria (ca. 24%), Verrucomicrobia (6%), Bacteroidetes (6%) and other phyla. These distinct genomic and molecular differences suggest distinctly different aggregate compositions of bacteria that produce 3-OH-FAs in the marine realm vs soils. Moreover, we find that the ratio of anteiso to normal C13 3-OH-FA (RAN13) measured in surface sediments is highly correlated with annual mean SST throughout the temperature transect. When applied to a short sediment core from the East China Sea, the SST changes reconstructed by the RAN13 proxy are comparable to instrumental SST data. These findings demonstrate that RAN13 and potentially other, so far undiscovered, proxies based on 3-OH-FAs have potential for environmental and palaeoclimate applications in marine environments.